Transcriptional responses of human intestinal epithelial HT-29 cells to spore-displayed p40 derived from Lacticaseibacillus rhamnosus GG.

BACKGROUNDS: The aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells. RESULTS: p40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action. CONCLUSIONS: This study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40.

Surface display of p75, a Lactobacillus rhamnosus GG derived protein, on Bacillus subtilis spores and its antibacterial activity against Listeria monocytogenes.

Lactobacillus rhamnosus p75 protein with peptidoglycan hydrolase (PGH) activity is one of the key molecules exhibiting anti-apoptotic and cell-protective activity for human intestinal epithelial cells. In this study, with the goal of developing new probiotics, the p75 protein was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchoring motif. The PGH activity, stability, and the antibacterial activity of the spore-displayed p75 (CotG-p75) protein were also investigated. The PGH activity of the CotG-p75 against peptidoglycan extracted from B. subtilis was confirmed by the ninhydrin test. Under various harsh conditions, compared to the control groups, the PGH activities of CotG-p75 were very stable in the range of pH 3-7 and maintained at 70% at 50 °C. In addition, the antibacterial activity of CotG-p75 against Listeria monocytogenes was evaluated by a time-kill assay. After 6 h incubation in phosphate-buffered saline, CotG-p75 reduced the number of viable cells of L. monocytogenes by up to 2.0 log. Scanning electron microscopy analysis showed that the cell wall of L. monocytogenes was partially damaged by the treatment with CotG-p75. Our preliminary results show that CotG-p75 could be a good candidate for further research to develop new genetically engineered probiotics.

KL1333, a Novel NAD+ Modulator, Improves Energy Metabolism and Mitochondrial Dysfunction in MELAS Fibroblasts.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), one of the most common maternally inherited mitochondrial diseases, is caused by mitochondrial DNA mutations that lead to mitochondrial dysfunction. Several treatment options exist, including supplementation with CoQ10, vitamins, and nutrients, but no treatment with proven efficacy is currently available. In this study, we investigated the effects of a novel NAD+ modulator, KL1333, in human fibroblasts derived from a human patient with MELAS. KL1333 is an orally available, small organic molecule that reacts with NAD(P)H:quinone oxidoreductase 1 (NQO1) as a substrate, resulting in increases in intracellular NAD+ levels via NADH oxidation. To elucidate the mechanism of action of KL1333, we used C2C12 myoblasts, L6 myoblasts, and MELAS fibroblasts. Elevated NAD+ levels induced by KL1333 triggered the activation of SIRT1 and AMPK, and subsequently activated PGC-1α in these cells. In MELAS fibroblasts, KL1333 increased ATP levels and decreased lactate and ROS levels, which are often dysregulated in this disease. In addition, mitochondrial functional analyses revealed that KL1333 increased mitochondrial mass, membrane potential, and oxidative capacity. These results indicate that KL1333 improves mitochondrial biogenesis and function, and thus represents a promising therapeutic agent for the treatment of MELAS.